Methods for determining adjuvanted protein concentration and percent adsorption using intrinsic fluorescence
Inventors
AUSAR, Salvador Fernando • ROQUE, Cristopher • STRAHLENDORF, Kirsten April • Rahman, Nausheen
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12320753-B2
Publication Date
2025-06-03
Expiration Date
Abstract
Methods and systems for determining the concentration of compounds, such as proteins, in compositions comprising adjuvanted complexes of the compounds are provided. The methods and systems generally comprise (i) measuring the intrinsic fluorescence of a composition comprising adjuvanted complexes of the compound and (ii) comparing the measured fluorescence intensity value to a calibration curve prepared using known concentrations of the compound. Also provided are methods and systems for determining percent adsorption of a selected compound to an adjuvant.
Core Innovation
The invention relates to a method for determining the concentration of a selected compound in a composition comprising an adjuvanted complex of the selected compound. The selected compound includes at least one aromatic amino acid, and the method obtains a fluorescence intensity value by exciting the composition at a wavelength of between 250 and 300 nm and determining an emission spectrum at a wavelength of between 300 and 500 nm to obtain the fluorescence intensity value.
The method compares the fluorescence intensity value with a calibration curve prepared using fluorescence values of at least three calibration samples comprising different known concentrations of a representative compound that is representative of the selected compound. The comparison with the calibration curve thereby determines the concentration of the selected compound in the composition comprising the adjuvanted complex of the selected compound.
Intrinsic fluorescence without added fluorescing probe or marker is used for nondestructively determining concentration and related adsorption behavior in vaccine compositions containing adjuvanted complexes. The disclosure further discusses determining % adsorption by comparing concentrations in an initially soluble sample versus supernatant after settling of adjuvant complexes, and a process analytical technology-oriented measurement approach including fluorescence flow cells and photometers for in-line proof-of-concept measurements.
Claims Coverage
The independent claim covers a fluorescence-based concentration determination of a selected compound in an adjuvanted-complex composition by using intrinsic fluorescence excitation and emission and a calibration curve prepared from representative compound standards. The inventive features are further refined by dependent claims that specify aromatic amino acid identity, calibration-point schemes, intrinsic measurement without probes, representative-compound relationships, adjuvant component types, and optional measurement formats.
Excitation and emission fluorescence intensity for adjuvanted complexes
Exciting a composition comprising an adjuvanted complex of a selected compound that includes at least one aromatic amino acid at a wavelength of between 250 and 300 nm, and determining an emission spectrum at a wavelength of between 300 and 500 nm to obtain the fluorescence intensity value for the composition.
Calibration curve using representative compound standards
Comparing the fluorescence intensity value obtained with a calibration curve prepared using fluorescence values of at least three calibration samples comprising different known concentrations of a representative compound that is representative of the selected compound, to thereby determine the concentration of the selected compound in the composition comprising the adjuvanted complex of the selected compound.
Across the independent claim, the core claim coverage is centered on measuring intrinsic fluorescence via defined excitation and emission ranges for a selected compound in an adjuvanted complex, and determining concentration by comparison to a calibration curve generated from fluorescence of at least three representative-compound calibration samples. Dependent claims narrow and refine this framework by adding aromatic amino acid identity, calibration construction constraints, intrinsic operation, and examples of adjuvant or aluminum-salt composition and measurement format.
Stated Advantages
Nondestructively determining concentration of proteins or other aromatic-amino-acid-containing selected compounds in vaccine compositions containing adjuvanted complexes.
Determining % adsorption by comparing concentrations in an initially soluble sample versus supernatant after settling of adjuvant complexes.
Supporting PAT-oriented inline/on-line/off-line fluorescence measurement using fluorescence flow cells and photometers.
Minimal interference from aluminum salt adjuvants (AlOOH/AlPO4) is discussed in the supporting experimental examples.
Documented Applications
Determining the concentration of a selected compound in vaccine compositions comprising adjuvanted complexes.
Estimating % adsorption of adjuvant complexes by comparing concentrations in a initially soluble sample versus a supernatant after settling.
Process analytical technology measurement approaches for in-line fluorescence flow-cell measurements, including proof-of-concept disposable flow-cell measurements and comparisons to micro-Kjeldahl.
Interested in licensing this patent?